Oligonucleotides for modulating apolipoprotein E (APOE) expression and methods of use thereof

Abstract

The present disclosure relates to oligonucleotides, particularly antisense oligonucleotides (ASOs) and pharmaceutically acceptable salts thereof, that can hybridize to APOE pre-mRNA or mRNA and reduce its expression. The ASOs disclosed herein can reduce translation of APOE protein in mammals (e.g., humans). The present disclosure further relates to a method for treating a disease or disorder in a subject in need thereof by administering the antisense oligonucleotides disclosed herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 484,581, filed February 13, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence table XML reference This application contains a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file, created on February 13, 2024, has the file name 767173_000017_SL.xml and is 403,882 bytes in size. [Background technology]

[0003] Neurodegenerative diseases are caused by the progressive loss of neuronal structure or cellular function and subsequent cell death in neuronal tissue, a process known as neurodegeneration. Neurodegeneration can progress through various components or levels of neural circuits, resulting in characteristic clinical symptoms such as Alzheimer's disease, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, prion diseases, and other human diseases.

[0004] As a non-limiting example, Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in elderly individuals. Senile plaques composed of amyloid beta (Aβ) fibrils, neurofibrillary tangles composed of aggregated tau protein, and neuronal dysplasia and neuronal loss characterize AD pathology and constitute the diagnostic biomarker profile for AD (see Jack CR, Jr. et al. (2018) Alzheimers Dement 14:535-562). The earliest pathological changes may occur more than 20 years before the onset of clinical symptoms of AD (see Jack CR, Jr. et al. Alzheimer's Disease Neuroimaging I (2009) Brain 132:1355-1365; Braak H, et al. (2011) J Neuropathol Exp Neurol 70:960-969; Bateman RJ et al. (2012) N Engl J Med 367:795-804; Gordon BA et al. (2018) Lancet Neurol 17:241-250; Barthelemy NR et al. (2020) Nat Med 26:398-407; Quiroz YT et al. (2020) Lancet Neurol 19:513-521), suggesting opportunities for early intervention and disease-modifying therapies.

[0005] AD susceptibility is influenced by common genetic variants, among which the APOE allele variant ε4 (APOE4) stands out due to its large effect size and significant enrichment within patient populations. Compared to the risk-neutral ε3 / ε3 genotype, inheritance of one or two ε4 alleles increases AD risk by 3-4-fold or 9-15-fold, respectively (see Farrer LA, et al. (1997) JAMA 278:1349-1356; Genin E, et al. (2011) Mol Psychiatry 16:903-907; Neu SC, et al. (2017) JAMA Neurol 74:1178-1189). Furthermore, compared with ε4 noncarriers, inheritance of one ε4 allele decreases the age at onset of AD by 2-8 years, and inheritance of two ε4 alleles decreases the age at onset by 5-16 years (see Corder EH, et al. (1993) Science 261:921-923. Sando SB, et al. (2008) BMC Neurol 8:9). In a meta-analysis of 38 paired case-control samples across multiple ethnic groups, the mean allele frequency of ε4 was found to be 38% in AD patients compared with 14% in cognitively healthy individuals (Alzgene. APOE_2 / 3 / 4. http: / / www.alzgene.org / meta.asp?geneID=83, updated 2010).

[0006] The human APOE protein consists of a receptor-binding domain in the amino-terminal domain and a lipid-binding domain in the carboxy-terminal domain. Nascent APOE protein is secreted by cells and associates with the cell surface ATP-binding cassette transporters ABCA1 and ABCG1. This allows cholesterol and phospholipids to be transferred to nascent APOE to form lipoprotein particles (see Wahrle SE, et al. (2004) J Biol Chem 279:40987-40993).

[0007] In the healthy brain, APOE is primarily expressed in astrocytes, microglia, vascular wall cells, and choroid plexus cells; however, under pathological or injury conditions, APOE expression is significantly upregulated in myeloid cells, including microglia and macrophages (see Xu Q, et al. (2006) J Neurosci 26:4985-4994; Yamazaki et al. (2019) Nat Rev Neurol 15(9):501-518). APOE plays physiological and pathophysiological roles, shuttling cholesterol and other lipids between various brain cell types via cell surface APOE receptors (see Hauser PS, et al. (2011) Prog Lipid Res 50:62-74; Yamazaki et al. (2019) Nat Rev Neurol 15(9):501-518).

[0008] The common APOE ε4 and ε3 allele variants encode the APOE4 and APOE3 protein isoforms, respectively. APOE4 differs from APOE3 at amino acid position 112 (APOE3: Cys112, APOE4: Arg112). Despite this difference in only a single amino acid substitution, APOE4 exerts numerous effects on the pathogenesis of AD (see Yamazaki Y, et al. (2019) Nat Rev Neurol 15:501-518). This is often characterized as a toxic gain-of-function APOE effect. The most prominent feature of such a toxic gain-of-function effect is that APOE4 reduces Aβ clearance and promotes Aβ aggregation, which correlates with the clinical phenotype of accelerated Aβ plaque deposition in ε4 allele carriers, with deposition most accelerated in ε4 / ε4 homozygous carriers. In both humans and animal models, APOE4 is associated with loss of blood-brain barrier (BBB) ​​integrity (see Ishii M and Iadecola C (2020) Nature 581:31-32; Montagne A et al. (2020) Nature 581:71-76; Jackson RJ, et al. (2022) Brain 145:3582-3593). Additionally, the ε4 allele is also associated with high levels of TAR DNA-binding protein 43 (TDP-43) pathology in the brains of individuals with AD (see Vossel KA, et al. (2013) Neurocase 19:295-301; Josephs KA, et al. (2017) Lancet Neurol 16:917-924; Yang HS, et al. (2018) Lancet Neurol 17:773-781).Furthermore, the ε4 allele increases the risk of developing Lewy body dementia (see Tsuang D et al. (2013) JAMA Neurol 70:223-228, Bras J et al. (2014) Hum Mol Genet 23:6139-6146, Guerreiro R et al. (2018) Lancet Neurol 17:64-74) and Parkinson's disease dementia (PDD) (see Huang X, et al. (2006) Arch Neurol 63:189-193, Irwin DJ, et al. (2012) Ann Neurol 72:587-598, Tsuang D et al. (2013) JAMA Neurol 70:223-228, Tropea TF, et al. (2018) Mov Disord 33:289-297).

[0009] Experimental studies have shown that APOE4 exacerbates tau pathology and tau-mediated neurodegeneration in animal models. Furthermore, APOE4 induces effects that can be characterized as the decline in physiological and pathophysiological functions of APOE, including microglial responsiveness, neuroinflammation, energy metabolism, cholesterol and lipid transport and homeostasis, synaptic integrity and plasticity, glucose metabolism, and neurovascular integrity and function (see Liu CC, et al. Nat Rev Neurol 9:106-118; Yamazaki Y, et al. (2019) Nat Rev Neurol 15:501-518; Martens YA, et al. (2022) Neuron 110:1304-1317). Furthermore, APOE4 disrupts physiological functions of APOE, such as microglial responsiveness, neuroinflammation, energy metabolism, cholesterol and lipid transport and homeostasis, synaptic integrity and plasticity, glucose metabolism, and neurovascular integrity and function (see Liu CC, et al. Nat Rev Neurol 9:106-118; Yamazaki Y, et al. (2019) Nat Rev Neurol 15:501-518; Martens YA, et al. (2022) Neuron 110:1304-1317).

[0010] Preclinical data are available demonstrating that certain APOE-targeted ASOs administered via intracerebroventricular (icv) injection early in the pathological onset of AD in mouse models can be effective in reducing brain amyloid burden. Most importantly, this intervention prevents the development of tau pathology in AD mice, reduces neurite malformation, and reduces levels of neurofilament light chain (NfL), a marker of neuronal damage, regardless of amyloid pathology or the timing of ASO administration relative to the onset of AD pathology. Additional readouts confirmed that icv delivery of APOE ASO prevented tau pathology and associated neurodegeneration, maintained synaptic density (reduced synaptic loss), and reduced the release of neuroinflammatory markers and pro-inflammatory cytokines in the brain, without affecting lipid levels in the liver and plasma (see Huynh TV, et al. (2017) Neuron 96:1013-1023 e1014; Litvinchuk A. et al. (2021) Ann Neurol 89:952-966). Recent data utilizing biochemical in vitro and in vivo methodologies in rodent disease models suggest that certain APOE-targeting monoclonal antibodies (α-APOE antibodies), which are selective for non-lipidated aggregated APOE, can preferentially bind to APOE in amyloid plaques. These antibodies have a similar mode of action and efficacy compared to Aβ-targeting monoclonal antibodies (anti-Aβ antibodies), which also bind to Aβ aggregates and reduce Aβ aggregates via a microglial-mediated clearance mechanism (see Liao F et al. (2018) J Clin Invest 128:2144-2155). Furthermore, certain α-APOE antibodies appear to specifically bind to dense-core plaques, while other anti-Aβ antibodies bind to widespread amyloid, including that deposited in cerebral blood vessels in cerebral amyloid angiopathy (CAA). Mechanistically, α-APOE antibodies specifically targeting plaques initially enhance glial activation, thereby persistently recruiting microglia and improving vascular function, whereas less specific anti-Aβ antibodies targeting vascular amyloid induce chronic perivascular astrogliosis and persistence of CAA pathology (see Xiong M, et al. (2021) Sci Transl Med 13, 581). The resulting neurovascular pathology and damage observed in animal models is consistent with amyloid-associated imaging abnormalities (ARIA-E & ARIA-H pathology) observed in human subjects treated with anti-Aβ antibodies (see Sperling RA et al. (2011) Alzheimers Dement 7:367-385, 2011; Salloway S, et al. (2022) JAMA Neurol 79:13-21). A related dataset demonstrates that after sustained treatment with α-APOE antibodies in AD mouse models, tau seeding, spreading, and resulting dysplasia in neurites is reduced, consistent with a reduction in overall individual amyloid burden (see Gratuze M, Jiang H, Wang C, Xiong M, Bao X, Holtzman DM (2022) Ann Neurol 91:847-852).Although the efficacy of amyloid- and APOE-targeted molecules in reducing amyloid plaques is similar, APOE-targeted molecules appear to offer an advantage by mediating additional beneficial effects on vascular phenotype while also reducing tau seeding, nucleic acid pathology, and tau markers (see Liao F et al. (2018) J Clin Invest 128:2144-2155; Xiong M, et al. (2021) Sci Transl Med 13,581; Gratuze M, et al. (2022) Ann Neurol 91:847-852). Unfortunately, antibody therapies, such as α-APOE antibodies, are limited to extracellular effects due to the potential for insufficient penetration into brain tissue due to the antibody's large molecular weight, short retention in the brain after direct administration, and short duration of effect. Therefore, additional agents and methods for reducing APOE expression are needed. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Jack CR, Jr. et al. (2018) Alzheimers Dement 14:535-562 [Non-patent document 2] Jack CR, Jr. et al.Alzheimer's Disease Neuroimaging I(2009)Brain 132:1355-1365 [Non-patent document 3] Braak H, et al. (2011) J Neuropathol Exp Neurol 70:960-969 [Non-patent document 4] Bateman RJ et al. (2012)N Engl J Med 367:795-804 [Non-Patent Document 5] Gordon BA et al. (2018) Lancet Neurol 17:241-250 [Non-patent document 6] Barthelemy NR et al. (2020) Nat Med 26:398-407 [Non-Patent Document 7] Quiroz YT et al. (2020) Lancet Neurol 19:513-521 [Non-patent document 8] Farrer LA, et al. (1997) JAMA 278:1349-1356 [Non-Patent Document 9] Genin E, et al. (2011) Mol Psychiatry 16:903-907 [Non-Patent Document 10] Neu SC, et al. (2017) JAMA Neurol 74:1178-1189 [Non-Patent Document 11] Corder EH, et al. (1993) Science 261:921-923 [Non-Patent Document 12] Sando SB, et al. (2008) BMC Neurol 8:9 [Non-Patent Document 13] Alzgene. APOE_2 / 3 / 4. http: / / www.alzgene.org / meta.asp?geneID=83, updated 2010 [Non-Patent Document 14] Wahrle SE, et al. (2004) J Biol Chem 279:40987-40993 Summary of the Invention

[0012] [Claim Summary] As described herein, the present disclosure relates to antisense oligonucleotides and methods of use thereof. Accordingly, in one aspect, the present disclosure provides an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, comprising a sequence at least 80% identical to SEQ ID NOs: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81.

[0013] In another aspect, the disclosure provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a sequence selected from the group consisting of SEQ ID NOs: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, and 77-81. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof has a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, and 81.

[0014] In another aspect, the disclosure provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a sequence at least 80% identical to SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, or 229.

[0015] In another aspect, the disclosure provides an antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a sequence selected from the group consisting of SEQ ID NOs: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof has a sequence selected from the group consisting of SEQ ID NOs: 127, 132, 147, 202, and 220.

[0016] In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 15 to about 25 nucleobases. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 18 to about 22 nucleobases. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 20 nucleobases. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 18 nucleobases.

[0017] In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises from about 12 to about 25 nucleobases. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises from about 14 to about 18 nucleobases. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 16 nucleobases.

[0018] In certain embodiments, antisense oligonucleotide comprises one or more modified internucleoside linkages.Modified internucleoside linkages can be selected from the group consisting of phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesylphosphoramidate, and combinations thereof.In certain embodiments, antisense oligonucleotide comprises one or more phosphorothioate internucleoside linkages.In certain embodiments, all of the internucleoside linkages are phosphorothioate linkages.

[0019] In certain embodiments, the antisense oligonucleotide comprises DNA. In certain embodiments, the antisense oligonucleotide comprises RNA. In certain embodiments, the antisense oligonucleotide comprises a protein and / or polypeptide. In certain embodiments, the antisense oligonucleotide comprises a small molecule.

[0020] In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleosides. The modified nucleosides may be selected from the group consisting of 2'-modified nucleosides, 4'-modified nucleosides, bridged nucleosides, phosphorodiamidate morpholines, locked nucleic acids, ethylene-bridged nucleic acids, glycol nucleic acids, hexitol nucleic acids, cyclohexene nucleic acids, arabinonucleic acids, peptide nucleic acids, threose nucleic acids, tricyclo-2'-deoxy-nucleotides, 1'-deoxyribopentose, 1',2'-dideoxyribopentose, 2',3'-dideoxyribopentose, 2',3'-didehydro-2',3'-dideoxyribopentose, non-locked nucleic acids, and combinations thereof. The antisense oligonucleotide may comprise one or more 2'-nucleoside modifications. The antisense oligonucleotide may comprise at least eight 2'-nucleoside modifications. The antisense oligonucleotide may contain at least 10 2'-nucleoside modifications.

[0021] In certain embodiments, all nucleosides of the antisense oligonucleotide comprise a 2'-nucleoside modification, which may be selected from the group consisting of 2'-fluoro-nucleosides, 2'-O-methyl-nucleosides, 2'-O-methoxyethyl nucleosides, 2'-O-benzyl-2'-deoxynucleosides, 2'-O-methyl-4-pyridinyl nucleosides (2'-O-CH2Py(4)), 2'-amino-nucleosides, and combinations thereof.

[0022] In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleobases.The modified nucleobases can be methylated nucleobases.The methylated nucleobases can be selected from the group consisting of 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof.The modified nucleobases can be acetylated nucleobases.The acetylated nucleobases can be N4-acetylcytosine. The modified nucleobase may be selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole modified adenine, N-ethylpiperidine-6-triazole modified adenine, 6-phenylpyrrolo-cytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine, and combinations thereof.

[0023] In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises an inverted nucleotide.

[0024] In certain embodiments, the antisense oligonucleotide is a gapmer. Gapmers can contain flanking RNA nucleotides at the 5'-terminus, the 3'-terminus, or both. The flanking RNA nucleotides at the 5'-terminus and / or the 3'-terminus can independently range from 1 nucleotide to about 9 nucleotides. The length of a gapmer can range from about 18 to about 22 nucleosides. The length of a gapmer can be 18 nucleosides. The length of a gapmer can be 10 central 2'-deoxynucleosides and 4 flanking RNA nucleotides at both the 5'-terminus and the 3'-terminus. The length of a gapmer can be 20 nucleosides. The length of a gapmer can be 10 central 2'-deoxynucleosides and / or 5 flanking ribonucleosides at both the 5'-terminus and the 3'-terminus.

[0025] In certain embodiments, gapmers range in length from about 14 to about 22 nucleosides. The flanking nucleotides at the 5' and / or 3' ends independently range from about 1 to about 5 nucleotides. Gapmers can contain 10 central 2'-deoxynucleotides and / or 3 flanking RNA nucleotides at both the 5' and 3' ends. Gapmers can be 16 nucleosides in length.

[0026] In certain embodiments, administration of an antisense oligonucleotide to a subject reduces human APOE mRNA, pre-mRNA, protein expression, or a combination thereof. Human APOE pre-mRNA or mRNA may be reduced by about 10% or more. Human APOE pre-mRNA or mRNA may be reduced by about 30% or more. Human APOE pre-mRNA or mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 25% or more. Human APOE mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 70% or more. Human APOE protein may be reduced by about 25% or more. Human APOE protein may be reduced by about 50% or more. Human APOE protein may be reduced by about 70% or more.

[0027] In certain embodiments, the antisense oligonucleotide can hybridize to human APOE mRNA. The human APOE mRNA can comprise a sequence at least 80% identical to SEQ ID NO: 1. The human APOE mRNA can encode a protein comprising a sequence 85% identical to SEQ ID NO: 82. The APOE can be an isoform selected from the group consisting of APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, and APOE 7. Hybridization can be by Watson-Crick base pairing.

[0028] In certain embodiments, the antisense oligonucleotide may hybridize to human APOE pre-mRNA. The human APOE pre-mRNA may comprise a sequence at least 80% identical to SEQ ID NO: 230. The human APOE mRNA may encode a protein comprising a sequence 85% identical to SEQ ID NO: 82. The APOE may be an isoform selected from the group consisting of APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, and APOE 7. Hybridization may be by Watson-Crick base pairing.

[0029] In another aspect, the present disclosure relates to a method of treating a disease or disorder in a human subject in need thereof, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof.

[0030] In another aspect, the present disclosure relates to a method for treating a neurodegenerative disease, comprising administering to a subject in need thereof an antisense oligonucleotide described herein or a pharmaceutically acceptable salt thereof. The neurodegenerative disease may be selected from the group consisting of, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion disease, and combinations thereof. The neurodegenerative disease may be Alzheimer's disease. The neurodegenerative disease may be a tauopathy. The tauopathy may be selected from the group consisting of, but is not limited to, primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

[0031] In another aspect, the present disclosure relates to a method of treating acute or chronic injury, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof. The acute or chronic injury may be selected from the group consisting of, but is not limited to, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemobrain, and combinations thereof.

[0032] In another aspect, the present disclosure relates to a method of treating an acute or chronic inflammatory condition, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof. The acute or chronic inflammatory condition may be selected from the group consisting of, but not limited to, stroke, multiple sclerosis, infectious disease, immune response to the development of cancer, and combinations thereof.

[0033] In another aspect, the present disclosure relates to a method of treating a vascular disease or pathological vascular condition, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof. The vascular disease or pathological vascular condition may be selected from the group consisting of, but not limited to, cerebral microangiopathy, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, and combinations thereof.

[0034] In another aspect, the present disclosure relates to a method of treating a lipid storage disorder, comprising administering to a subject in need thereof an antisense oligonucleotide described herein or a pharmaceutically acceptable salt thereof. The lipid storage disorder may be selected from the group consisting of, but not limited to, cerebrotendinous xanthomatosis, Farber disease, Fabry disease, fucosidosis, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis AB variant, Krabbe disease, metachromatic leukodystrophy, multiple sulfatase deficiency, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sandhoff disease, Schindler disease, Tay-Sachs disease, Wolman disease, and combinations thereof.

[0035] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof.

[0036] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the treatment of a disease or disorder in a human subject in need thereof.

[0037] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject in need thereof.

[0038] In another aspect, the present disclosure relates to the use of the antisense oligonucleotide or its pharmaceutically acceptable salt described herein for the treatment of a neurodegenerative disease in a subject in need thereof.The neurodegenerative disease may be selected from the group consisting of, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion disease, and combinations thereof.The neurodegenerative disease may be a tauopathy. The tauopathy may be selected from the group consisting of primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

[0039] In certain embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered locally to a subject in need thereof. Local administration can be intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

[0040] In certain embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need thereof. [Brief explanation of the drawings]

[0041] [Figure 1] These are phase-contrast microscopy images showing that some ASOs (e.g., S1038 (SEQ ID NO: 24)) cause abnormal morphological cell changes in HEP3B cells, while other ASOs cause morphology equivalent to that of untreated controls. All cells were cultured for 48 hours with or without ASO. (A) is a microscopy image showing HEP3B cells not treated with ASO ("untreated control"). (B) is a microscopy image showing that HEP3B cells treated with ASO S1038 (12.5 nM (SEQ ID NO: 24)) exhibit abnormal epithelial cell morphology (e.g., loss of confluency, dense pyknotic nuclei), indicating cytotoxicity and cell death, compared to untreated HEP3B cells. (C) is a microscopy image showing that HEP3B cells treated with ASO S1094 (12.5 nM (SEQ ID NO: 80)) exhibit morphology and confluency equivalent to that of untreated HEP3B cells. This indicates that ASO S1094 (SEQ ID NO: 80) is non-toxic to HEP3B cells. DETAILED DESCRIPTION OF THE INVENTION

[0042] Mutation and expression of apolipoprotein E (APOE) are highly correlated with human diseases, particularly neurological disorders (e.g., Alzheimer's disease (AD), dementia with Lewy bodies, and Parkinson's disease). AD susceptibility is influenced by common genetic variants, among which the APOE allelic variant ε4 (APOE4) stands out due to its large effect size and significant enrichment within patient populations. Compared to the risk-neutral ε3 / ε3 genotype, inheritance of one or two ε4 alleles increases AD risk by 3-4-fold or 9-15-fold, respectively (see Farrer LA, et al. (1997) JAMA 278:1349-1356; Genin E, et al. (2011) Mol Psychiatry 16:903-907; Neu SC, et al. (2017) JAMA Neurol 74:1178-1189). Therefore, APOE has been investigated as a potential target for treating neurological diseases and other diseases in which APOE may play a role in disease pathology.Unfortunately, antibody therapy such as α-APOE antibody may have poor penetration into brain tissue due to the large molecular weight of antibody, short retention in the brain after direct administration, and short duration of effect, and is limited to extracellular effects.Therefore, there is a need for additional drugs and methods for reducing APOE expression.

[0043] antisense oligonucleotides The present disclosure relates to antisense oligonucleotides and pharmaceutically relevant salts thereof that target (e.g., hybridize, Watson-Crick base pair, bind, reduce expression of) APOE mRNA and / or pre-mRNA. In certain embodiments of the present disclosure, the antisense oligonucleotide or pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may comprise at least one of SEQ ID NOs: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NOs: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, or 81.

[0044] In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 3-81. In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NOs: 3-81. In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, or 81.

[0045] The target APOE oligonucleotide sequence can be a pre-mRNA. A pre-mRNA is a pre-mRNA resulting from DNA transcription. After transcription, the pre-mRNA undergoes processing steps such as the addition of a 5'-cap, the addition of a polyA tail, and the removal of introns by splicing to generate a mature mRNA. The target pre-mRNA can be a pre-mRNA encoding any APOE isoform, such as APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s, or APOE 7. For example, the target can be an APOE pre-mRNA having a nucleic acid sequence according to SEQ ID NO: 230. In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof is selected from the group consisting of SEQ ID NOs: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 0, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, or 229. In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 84, 119, 120, 123, 124, 129, 133-136, 168, 185, 228, or 229. In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 84-229.In certain embodiments of the present disclosure, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises a sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of SEQ ID NOs: 127, 132, 147, 202, or 220. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may comprise at least one of SEQ ID NOs: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, or 229. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof may comprise at least one of SEQ ID NOs: 84, 119, 120, 123, 124, 129, 133-136, 168, 185, 228, or 229. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NOs: 127, 132, 147, 202, or 220. In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one of SEQ ID NOs: 84-229.

[0046] The antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprises about 15 to about 25 nucleobases, about 15 to about 24 nucleobases, about 15 to about 23 nucleobases, about 15 to about 22 nucleobases, about 15 to about 21 nucleobases, about 15 to about 20 nucleobases, about 15 to about 19 nucleobases, about 15 to about 18 nucleobases, about 18 to 22 nucleobases, about 18 to about 21 nucleobases, about 18 to about 20 nucleobases, about 15 nucleobases, about 16 nucleobases, about 17 nucleobases, about 18 nucleobases, about 19 nucleobases, about 20 nucleobases, about 21 nucleobases, or about 22 nucleobases. In some embodiments, the antisense oligonucleotide or pharmaceutically acceptable salt thereof is from about 12 to about 25 nucleobases, from about 13 to about 25 nucleobases, from about 14 to about 25 nucleobases, from about 12 to about 24 nucleobases, from about 12 to about 23 nucleobases, from about 12 to about 22 nucleobases, from about 12 to about 21 nucleobases, from about 12 to about 20 nucleobases, from about 12 to about 19 nucleobases, from about 12 to about 18 nucleobases, from about 12 to about 17 nucleobases, from about 12 to about 16 nucleobases, from about 12 to about 28 nucleobases, from about 12 to about 29 nucleobases, from about 12 to about 30 nucleobases, from about 12 to about 31 nucleobases, from about 12 to about 32 nucleobases, from about 12 to about 33 nucleobases, from about 12 to about 34 nucleobases, from about 12 to about 35 nucleobases, from about 12 to about 36 nucleobases, from about 12 to about 37 nucleobases, from about 12 to about 38 nucleobases, from about 12 to about 39 nucleobases, from about 12 to about 40 nucleobases, from about 12 to about 41 nucleobases, from about 12 to about 42 nucleobases, from about 12 to about 43 nucleobases, from about 12 to about 44 nucleobases, from about The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain about 20 nucleobases. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain about 18 nucleobases. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain about 16 nucleobases. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain about 16 nucleobases. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof may contain about 16 nucleobases.

[0047] In certain embodiments, antisense oligonucleotide can comprise one or more modified internucleoside bonds.Modified internucleoside bonds can be phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesylphosphoramidate or combinations thereof.Antisense oligonucleotide can comprise one or more phosphorothioate internucleoside bonds.In certain embodiments, all of internucleoside bonds are phosphorothioate bonds.

[0048] Antisense oligonucleotides can include DNA, RNA, proteins and / or polypeptides. In certain embodiments, antisense oligonucleotides include small molecules.

[0049] In certain embodiments, antisense oligonucleotide can comprise one or more modified nucleosides.Modified nucleosides can be 2'-modified nucleosides, 4'-modified nucleosides, bridged nucleosides, phosphorodiamidate morpholine, locked nucleic acid, ethylene-bridged nucleic acid, glycol nucleic acid, hexitol nucleic acid, cyclohexene nucleic acid, arabino nucleic acid, peptide nucleic acid, threose nucleic acid, tricyclo-2'-deoxy-nucleotide, 1'-deoxyribopentose, 1',2'-dideoxyribopentose, 2',3'-dideoxyribopentose, 2',3'-didehydro-2',3'-dideoxyribopentose, non-locked nucleic acid, or combinations thereof.Antisense oligonucleotide can comprise one or more 2'-nucleoside modifications. The antisense oligonucleotide may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-nucleoside modifications. The antisense oligonucleotide may contain at least 8 2'-nucleoside modifications. The antisense oligonucleotide may contain at least 10 2'-nucleoside modifications.

[0050] In certain embodiments, all nucleosides of the antisense oligonucleotide contain 2'-nucleoside modifications. The 2'-nucleoside modifications can be 2'-fluoro-nucleosides, 2'-O-methyl-nucleosides, 2'-O-methoxyethyl-nucleosides, 2'-O-benzyl-2'-deoxynucleosides, 2'-O-methyl-4-pyridinyl-nucleosides (2'-O-CH2Py(4)), 2'-amino-nucleosides, or combinations thereof.

[0051] In certain embodiments, antisense oligonucleotides can comprise one or more modified nucleobases (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleobases).Modified nucleobases can be methylated nucleobases.Modified nucleobases can be 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, or combinations thereof.Modified nucleobases can be acetylated nucleobases.Acetylated nucleobases can be N4-acetylcytosine. The modified nucleobase can be pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole-modified adenine, N-ethylpiperidine-6-triazole-modified adenine, 6-phenylpyrrolo-cytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine, or a combination thereof.

[0052] In certain embodiments, the antisense oligonucleotide or a pharmaceutically acceptable salt thereof further comprises an inverted nucleotide.

[0053] In certain embodiments, the antisense oligonucleotide is a gapmer. Gapmers can contain flanking RNA nucleotides at the 5'-terminus, the 3'-terminus, or both. The flanking RNA nucleotides at the 5'-terminus and / or the 3'-terminus can independently range from about 1 to about 10 nucleotides, about 1 to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, or about 1 to about 3 nucleotides. The length of a gapmer can range from about 18 to about 22 nucleosides (e.g., about 18, 19, 20, 21, or 22 nucleosides). The length of a gapmer can be 18 nucleosides. A gapmer can contain about 10 central 2'-deoxynucleosides and about 4 flanking RNA nucleotides at both the 5'-terminus and the 3'-terminus. The length of a gapmer can be 20 nucleosides. A gapmer can contain about 10 central 2'-deoxynucleosides and / or about 5 flanking ribonucleosides at both the 5' and 3' ends.

[0054] Gapmers can be of any length. The length of the gapmer nucleotides or nucleosides can be about 14 to about 22, about 15 to about 22, about 16 to about 22, about 17 to about 22, about 18 to about 22, about 19 to about 22, about 20 to about 22, about 21 to about 22, about 14 to about 21, about 14 to about 20, about 14 to about 19, about 14 to about 18, about 14 to about 17, about 14 to about 16, or about 14 to about 15. The length of the gapmer nucleotides or nucleosides can be about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, or about 22. Gapmers can contain about 10 central 2'-deoxynucleosides and about 3 flanking RNA nucleotides at both the 5' and 3' ends. A gapmer can contain about 10 central 2'-deoxynucleosides and / or about 3 flanking RNA nucleotides at both the 5' and 3' ends. In certain embodiments, the gapmer is 16 nucleosides in length. In certain embodiments, the gapmer is 18 nucleosides in length. In certain embodiments, the gapmer ranges in length from about 14 to about 22 nucleosides. In certain embodiments, the flanking RNA nucleotides at the 5' and / or 3' ends, independently, range from about 1 nucleotide to about 5 nucleotides.

[0055] Gapmers can have any combination of nucleotide lengths and numbers of flanking nucleotides (e.g., 3'-terminal flanking nucleotides, 5'-terminal flanking nucleotides, or both) described herein. The flanking nucleotides (3'-terminal flanking nucleotides, 5'-terminal flanking nucleotides) can contain any number of modified nucleotides or nucleosides. The modified nucleosides or nucleotides can be modified by any means, such as any of the modifications described herein (e.g., 2'-modifications, locked nucleic acid modifications). The modified nucleosides or nucleotides within a flanking region can be contiguous. The modified nucleosides or nucleotides within a flanking region can be non-contiguous (e.g., an unmodified nucleotide can be present between two or more modified nucleotides in a flanking region). A gapmer can contain 14 nucleotides, with the 5' and / or 3' ends independently containing about 1 to about 10 nucleotides, about 1 to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides. For example, a gapmer can be 1-12-1, 1-11-2, 2-11-1, 3-10-1, 1-10-3, 2-10-2, 3-9-2, 2-9-3, 1-9-4, 4-9-1, 5-8-1, 1-8-5, 4-8-2, 2-8-4, or 3-8-3, where the values ​​X, Y, and Z indicate the 5'-adjacent nucleotide, the central nucleotide, and the 3-adjacent nucleotide. A gapmer can contain 15 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides.For example, the gapmer can be 1-13-1, 1-12-2, 2-12-1, 3-11-1, 1-11-3, 2-11-2, 3-10-2, 2-10-3, 1-10-4, 4-10-1, 5-9-1, 1-9-5, 4-9-2, 2-9-4, 3-9-3, 4-8-3, 3-8-4, 2-8-5, 5-8-2, 1-8-6, or 6-8-1. A gapmer can contain 16 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides. For example, the gapmer can be 1-14-1, 1-13-2, 2-13-1, 3-12-1, 1-12-3, 2-12-2, 3-11-2, 2-11-3, 1-11-4, 4-11-1, 5-10-1, 1-10-5, 4-10-2, 2-10-4, 3-10-3, 4-9-3, 3-9-4, 2-9-5, 5-9-2, 1-9-6, 6-9-1, 7-8-1, 1-8-7, 6-8-2, 2-8-6, 5-8-3, 3-8-5, or 4-8-4. A gapmer can contain 17 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides. For example, the gapmer can be 1-15-1, 1-14-2, 2-14-1, 3-13-1, 1-13-3, 2-13-2, 3-12-2, 2-12-3, 1-12-4, 4-12-1, 5-11-1, 1-11-5, 4-11-2, 2-11-4, 3-11-3, 4-10-3, 3-10-4, 2-10-5, 5-10-2, 1-10-6, 6-10-1, 7-9-1, 1-9-7, 6-9-2, 2-9-6, 5-9-3, 3-9-5, 4-9-4, 8-8-1, 1-8-8, 7-8-2, 2-8-7, 6-8-3, 3-8-6, 5-8-4, or 4-8-5.A gapmer can contain 18 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides. For example, gapmers include 1-16-1, 1-15-2, 2-15-1, 3-14-1, 1-14-3, 2-14-2, 3-13-2, 2-13-3, 1-13-4, 4-13-1, 5-12-1, 1-12-5, 4-12-2, 2-12-4, 3-12-3, 4-11-3, 3-11-4, 2-11-5, 5-11-2, 1-11-6, 6-11-1, It can be 7-10-1, 1-10-7, 6-10-2, 2-10-6, 5-10-3, 3-10-5, 4-10-4, 8-9-1, 1-9-8, 7-9-2, 2-9-7, 6-9-3, 3-9-6, 5-9-4, 4-9-5, 9-8-1, 1-8-9, 8-8-2, 2-8-8, 7-8-3, 3-8-7, 6-8-4, 4-8-6, or 5-8-5. A gapmer can contain 19 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides.For example, gapmers include 1-17-1, 1-16-2, 2-16-1, 3-15-1, 1-15-3, 2-15-2, 3-14-2, 2-14-3, 1-14-4, 4-14-1, 5-13-1, 1-13-5, 4-13-2, 2-13-4, 3-13-3, 4-12-3, 3-12-4, 2-12-5, 5-12-2, 1-12-6, 6-12-1, 7-11-1, 1-11-7, 6-11-2, 2-11-6, 5-11-3, It can be 3-11-5, 4-11-4, 8-10-1, 1-10-8, 7-10-2, 2-10-7, 6-10-3, 3-10-6, 5-10-4, 4-10-5, 9-9-1, 1-9-9, 8-9-2, 2-9-8, 7-9-3, 3-9-7, 6-9-4, 4-9-6, 5-9-5, 10-8-1, 1-8-10, 9-8-2, 2-8-9, 8-8-3, 3-8-8, 7-8-4, 4-8-7, 5-8-6, or 6-8-5. A gapmer can contain 20 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides.For example, gapmers include 1-18-1, 1-17-2, 2-17-1, 3-16-1, 1-16-3, 2-16-2, 3-15-2, 2-15-3, 1-15-4, 4-15-1, 5-14-1, 1-14-5, 4-14-2, 2-14-4, 3-14-3, 4-13-3, 3-13-4, 2-13-5, 5-13-2, 1-13-6, 6-13-1, 7-12-1, 1-12-7, 6-12-2, 2-12-6, 5-12-3, 3-12-5, 4-12-4, 8-11-1, 1-11-8, 7-11-2, 2-11 -7, 6-11-3, 3-11-6, 5-11-4, 4-11-5, 9-10-1, 1-10-9, 8-10-2, 2-10-8, 7-10-3, 3-10-7, 6-10-4, 4-10-6, 5-10-5, 10-9-1, 1-9-10, 9-9-2, 2-9-9, 8-9-3, 3-9-8, 7-9-4, 4-9-7, 5-9-6, 6-9-5, 11-8-1, 1-8-11, 10-8-2, 2-8-10, 9-8-3, 3-8-9, 8-8-4, 4-8-8, 7-8-5, 5-8-7, or 6-8-6. A gapmer can contain 21 nucleotides, and the 5' end and / or the 3' end can independently contain from about 1 to about 10 nucleotides, from about 1 to about 9 nucleotides, from about 1 to about 8 nucleotides, from about 1 to about 7 nucleotides, from about 1 to about 6 nucleotides, from about 1 to about 5 nucleotides, from about 1 to about 4 nucleotides, from about 1 to about 3 nucleotides, or from about 1 to about 2 nucleotides.For example, gapmers include 1-19-1, 1-18-2, 2-18-1, 3-17-1, 1-17-3, 2-17-2, 3-16-2, 2-16-3, 1-16-4, 4-16-1, 5-15-1, 1-15-5, 4-15-2, 2-15-4, 3-15-3, 4-14-3, 3-14-4, 2-14-5, 5-14-2, 1-14-6, 6-14-1, 7-13-1, 1-13-7, 6-13-2, 2-13-6, 5-13-3, 3-13-5, 4-13-4, 8-12-1, 1-12-8, 7-12-2, 2-12-7, 6-12-3, 3-12-6, 5-12-4, 4-12-5, 9-11-1, 1-11-9 , 8-11-2, 2-11-8, 7-11-3, 3-11-7, 6-11-4, 4-11-6, 5-11-5, 10-10-1, 1-10-10, 9-10-2, 2-10-9, 8-10-3, 3-10-8, 7-10-4, 4-10-7, 5-10-6, 6-10-5, 11-9-1, 1-9-11, 10- The gapmer may be 9-2, 2-9-10, 9-9-3, 3-9-9, 8-9-4, 4-9-8, 7-9-5, 5-9-7, 6-9-6, 12-8-1, 1-8-12, 11-8-2, 2-8-11, 10-8-3, 3-8-10, 4-8-9, 9-8-4, 8-8-5, 5-8-8, 7-8-6, or 6-8-7. A gapmer may contain 22 nucleotides, and the 5' end and / or the 3' end may independently contain about 1 to about 10 nucleotides, about 1 to about 9 nucleotides, about 1 to about 8 nucleotides, about 1 to about 7 nucleotides, about 1 to about 6 nucleotides, about 1 to about 5 nucleotides, about 1 to about 4 nucleotides, about 1 to about 3 nucleotides, or about 1 to about 2 nucleotides.For example, gapmers are 1-20-1, 1-19-2, 2-19-1, 3-18-1, 1-18-3, 2-18-2, 3-17-2, 2-17-3, 1-17-4, 4-17-1, 5-16-1, 1-16-5, 4-16-2, 2-16-4, 3-16-3, 4-15-3, 3-15-4, 2-15-5, 5-15- 2, 1-15-6, 6-15-1, 7-14-1, 1-14-7, 6-14-2, 2-14-6, 5-14-3, 3-14-5, 4-14-4, 8-13-1, 1-13-8, 7-13-2, 2-13-7, 6-13-3, 3-13-6, 5-13-4, 4-13-5, 9-12-1, 1-12-9, 8-12-2, 2 -12-8, 7-12-3, 3-12-7, 6-12-4, 4-12-6, 5-12-5, 10-11-1, 1-11-10, 9-11-2, 2-11-9, 8-11-3, 3-11-8, 7-11-4, 4-11-7, 5-11-6, 6-11-5, 11-10-1, 1-10-11, 10-10-2, 2-10-1 0, 9-10-3, 3-10-9, 8-10-4, 4-10-8, 7-10-5, 5-10-7, 6-10-6, 12-9-1, 1-9-12, 11-9-2, 2-9-11, 10-9-3, 3-9-10, 4-9-9, 9-9-4, 8-9-5, 5-9-8, 7-9-6, 6-9-7, 13-8-1, 1-8-13,. It can be 12-8-2, 2-8-12, 11-8-3, 3-8-11, 4-8-10, 10-8-4, 9-8-5, 5-8-9, 8-8-6, 6-8-8, or 7-8-7.

[0056] In certain embodiments, administering antisense oligonucleotide to a subject reduces human APOE mRNA, pre-mRNA, protein expression, or a combination thereof.Human APOE mRNA can be reduced by about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.In certain embodiments, administering antisense oligonucleotide to a subject reduces human APOE mRNA, pre-mRNA, protein expression, or a combination thereof.Human APOE pre-mRNA or mRNA can be reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. Human APOE pre-mRNA may be reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. Human APOE pre-mRNA or mRNA may be reduced by about 10% or more. Human APOE pre-mRNA or mRNA may be reduced by about 30% or more. Human APOE pre-mRNA or mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 25% or more. Human APOE mRNA may be reduced by about 50% or more. Human APOE mRNA may be reduced by about 70% or more. Human APOE protein may be reduced by about 25% or more. Human APOE protein may be reduced by about 50% or more. Human APOE protein may be reduced by about 70% or more.

[0057] In certain embodiments, the antisense oligonucleotide can hybridize to human APOE mRNA. Human APOE mRNA can comprise a sequence that is at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. Human APOE mRNA can encode a protein that comprises a sequence that is approximately 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82. APOE can be the isoforms APOE 2, APOE 3, APOE 4, APOE 5f, APOE 5s and APOE 7. Hybridization can be by Watson-Crick base pairing.

[0058] In certain embodiments, antisense oligonucleotide can hybridize to human APOE pre-mRNA.Human APOE pre-mRNA can comprise at least 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence to SEQ ID NO:230.Human APOE pre-mRNA can encode a protein comprising about 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence to SEQ ID NO:82.APOE can be isoform APOE2, APOE3, APOE4, APOE5f, APOE5s or APOE7. Hybridization can be by Watson-Crick base pairing.

[0059] Antisense oligonucleotides can hybridize with APOE mRNA or APOE pre-mRNA containing single nucleotide polymorphisms (SNPs).Any single nucleotide polymorphism can be present in target APOE mRNA or APOE pre-mRNA (for example, the mRNA or pre-mRNA ASO described herein hybridizes), and single nucleotide polymorphisms include but are not limited to apoE3-R136S ("Christchurch" variant, apoE3-Ch), apoE3-V236E (apoE3-Jacksonville, apoE3-Jac), apoE4-R251G, or any combination thereof. In certain embodiments, the APOE mRNA or APOE pre-mRNA contains a single nucleotide polymorphism (SNP), and the SNP is selected from apoE3-R136S ("Christchurch" variant, apoE3-Ch), apoE3-V236E (apoE3-Jacksonville, apoE3-Jac), apoE4-R251G, or any combination of the foregoing.

[0060] Treatment method The present disclosure further relates to methods of using the oligonucleotides and pharmaceutically related salts disclosed herein for the treatment of diseases and disorders (e.g., human diseases and disorders, neurodegenerative diseases). The mode of action of oligonucleotides such as those described herein differs from the mode of action of antibodies or small molecules, and oligonucleotides can be used to treat, for example, They offer significant advantages in terms of (i) penetration into tissues, such as brain tissue, where their size is substantially smaller than that of antibodies, resulting in better penetration than antibodies and the potential for direct delivery to the brain (e.g., Noguchi Y, et al. (2017) MAbs 9:1210-1215, Pizzo ME, et al. (2018) J Physiol 596:445-475); (ii) retention in the brain and extended duration of effect after direct administration; (iii) blocking multiple target functions and activities, respectively; (iv) combination with other oligonucleotides or with antibodies or small molecules; and (v) inhibition of intracellular effects that are inaccessible to antibodies or cannot be inhibited via small molecules.

[0061] Taking AD or neurodegenerative diseases in general together, there is a clear need for effective therapeutic agents that can target the underlying pathobiological mechanisms. Counteracting the pathogenic gain-of-function toxic effects of APOE variants may be a therapeutic approach for patients with APOE ε4 who have been diagnosed with AD, DLB, PDD (see Li Y, Macyczko JR, Liu CC, Bu G (2022) Neurobiol Aging 115:20-28), or any other human patients or diseases associated with APOE dysregulation. Specifically, therapeutic effects can be achieved by suppressing the expression of mRNA encoding APOE. Alternatively, therapeutic effects can be achieved by suppressing the expression of pre-mRNA encoding APOE.

[0062] Two competing technologies have been primarily described for the specific suppression of mRNA expression: siRNA and antisense oligonucleotides (ASOs). siRNA targets mRNA in the cytoplasm. In contrast, RNase H-dependent ASOs target pre-mRNA in the nucleus. This allows ASOs to target intronic regions inaccessible to siRNA, significantly expanding the target space for ASOs. Furthermore, due to their double-stranded nature, siRNAs do not cross cell membranes by themselves, and a delivery system is required for their activity in vitro and in vivo. While there are siRNA delivery systems that efficiently deliver siRNA to liver cells in vivo, no system currently exists that can deliver siRNA outside liver tissue in vivo with sufficient efficacy. In contrast, modified ASOs can enter many cell types in vitro in sufficient quantities without transfection or delivery methods, resulting in potent and sequence-specific target knockdown. In vivo, naked, unconjugated ASOs achieve target knockdown in several different relevant tissues after systemic administration. Following direct administration, ASOs achieve potent and durable target knockdown in the central nervous system (CNS), indicating the strong potential of ASOs for the treatment of CNS diseases such as neurodegenerative disorders (see Geary RS et al. (2015) Adv Drug Deliv Rev 87:46-51).

[0063] In certain embodiments, the present disclosure relates to a method of treating a disease or disorder in a human subject in need thereof, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof.

[0064] In another aspect, the present disclosure relates to a method for treating a neurodegenerative disease, comprising administering to a subject in need thereof an antisense oligonucleotide described herein or a pharmaceutically acceptable salt thereof. The neurodegenerative disease can be Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion disease, or a combination thereof. The neurodegenerative disease can be Alzheimer's disease. The neurodegenerative disease can be a tauopathy. The tauopathy can be primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral frontotemporal dementia (Pick's disease), argyrophilic grain disease, or a combination thereof.

[0065] In another aspect, the present disclosure relates to a method of treating acute or chronic injury, comprising administering to a subject in need thereof an antisense oligonucleotide described herein or a pharmaceutically acceptable salt thereof. The acute or chronic injury can be any injury to the nervous system, including, but not limited to, diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, anoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemobrain, neuronal damage resulting from tumors, meningitis, encephalitis, and other inflammation in the body or nervous system, or a combination thereof.

[0066] In another aspect, the present disclosure relates to a method of treating an acute or chronic inflammatory condition in the nervous system or in a body extremity, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof. The acute or chronic inflammatory condition may be an autoimmune disease, multiple sclerosis, an infectious disease, an immune response to cancer, a tumor, or a combination thereof. The acute or chronic inflammatory condition may be an autoimmune disease, stroke, multiple sclerosis, an infectious disease, an immune response to cancer, a tumor, or a combination thereof.

[0067] In another aspect, the present disclosure relates to a method of treating a vascular disease or pathological vascular condition, comprising administering to a subject in need thereof an antisense oligonucleotide described herein, or a pharmaceutically acceptable salt thereof. The vascular disease or pathological vascular condition may be any human vascular disease, including, but not limited to, cerebrovascular disease, cerebral microangiopathy, diseases caused by or affected by blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular disease, peripheral arterial disease, carotid artery disease, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency thrombosis, deep vein thrombosis, or a combination thereof.

[0068] In another aspect, the present disclosure relates to a method of treating a lipid storage disorder, comprising administering to a subject in need thereof an antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described herein. The lipid storage disorder can be cerebrotendinous xanthomatosis, Farber disease, Fabry disease, fucosidosis, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis AB variant, Krabbe disease, metachromatic leukodystrophy, multiple sulfatase deficiency, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sandhoff disease, Schindler disease, Tay-Sachs disease, Wolman disease, or a combination thereof.

[0069] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof.

[0070] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the treatment of a disease or disorder in a human subject in need thereof.

[0071] In another aspect, the disclosure relates to the use of an antisense oligonucleotide, or a pharmaceutically acceptable salt thereof, described herein for the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject in need thereof.

[0072] In another aspect, the present disclosure relates to the use of the antisense oligonucleotide or its pharmaceutically acceptable salt described herein for the treatment of neurodegenerative disease in a subject in need thereof.Neurodegenerative disease can be Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion disease, or a combination thereof.Neurodegenerative disease can be Alzheimer's disease.Neurodegenerative disease can be tauopathy. The tauopathy can be primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral frontotemporal dementia (Pick's disease), argyrophilic grain disease, or a combination thereof.

[0073] In certain embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered locally to a subject in need thereof. Local administration can be intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

[0074] In certain embodiments, the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need thereof. [Example]

[0075] The following examples illustrate non-limiting embodiments of the present invention. The experiments below were performed in cells that endogenously express APOE; i.e., the cells do not represent artificial systems containing transfected reporter constructs. Such artificial systems generally result in higher levels of inhibition and lower IC50 values ​​than endogenous systems, which more closely resemble therapeutically relevant in vivo systems.

[0076] In certain embodiments, the present disclosure provides antisense oligonucleotide compounds that target human APOE nucleic acid.In certain embodiments, the human APOE nucleic acid is the APOE3 variant sequence shown in NCBI Reference Sequence NM_000041.4. 1 ctactcagcc ccagcggagg tgaaggacgt ccttccccag gagccgactg gccaatcaca 61 ggcaggaaga tgaaggttct gtgggctgcg ttgctggtca cattcctggc aggatgccag 121 gccaaggtgg agcaagcggt ggagacagag ccggagcccg agctgcgcca gcagaccgag 181 tggcagagcg gccagcgctg ggaactggca ctgggtcgct tttgggatta cctgcgctgg 241 gtgcagacac tgtctgagca ggtgcaggag gagctgctca gctcccaggt cacccaggaa 301 ctgagggcgc tgatggacga gaccatgaag gagttgaagg cctacaaatc ggaactggag 361 gaacaactga ccccggtggc ggaggagacg cgggcacggc tgtccaagga gctgcaggcg 421 gcgcaggccc ggctgggcgc ggacatggag gacgtgtgcg gccgcctggt gcagtaccgc 481 ggcgaggtgc aggccatgct cggccagagc accgaggagc tgcgggtgcg cctcgcctcc 541 cacctgcgca agctgcgtaa gcggctcctc cgcgatgccg atgacctgca gaagcgcctg 601 gcagtgtacc aggccggggc ccgcgagggc gccgagcgcg gcctcagcgc catccgcgag 661 cgcctggggc ccctggtgga acagggccgc gtgcgggccg ccactgtggg ctccctggcc 721 ggccagccgc tacaggagcg ggcccaggcc tggggcgagc ggctgcgcgc gcggatggag 781 gagatgggca gccggacccg cgaccgcctg gacgaggtga aggagcaggt ggcggaggtg 841 cgcgccaagc tggaggagca ggcccagcag atacgcctgc aggccgaggc cttccaggcc 901 cgcctcaaga gctggttcga gcccctggtg gaagacatgc agcgccagtg ggccgggctg 961 gtggagaagg tgcaggctgc cgtgggcacc agcgccgccc ctgtgcccag cgacaatcac 1021 tgaacgccga agcctgcagc catgcgaccc cacgccaccc cgtgcctcct gcctccgcgc 1081 agcctgcagc gggagaccct gtccccgccc cagccgtcct cctggggtgg accctagttt 1141 aataaagatt caccaagttt cacgca (SEQ ID NO: 1) MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDAD DLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH (SEQ ID NO: 82)

[0077] In certain embodiments, the present disclosure provides antisense oligonucleotide compounds that target human APOE pre-mRNA. An exemplary DNA sequence of APOE pre-mRNA is: 1 CTACTCAGCC CCAGCGGAGG TGAAGGACGT CCTTCCCCAG GAGCCGgtga gaagcgcagt 61 cgggggcacg gggatgagct caggggcctc tagaaagagc tgggaccctg ggaacccctg 121 gcctccaggt agtctcagga gagctactcg gggtcgggct tggggagagg aggagcgggg 181 gtgaggcaag cagcagggga ctggacctgg gaagggctgg gcagcagaga cgacccgacc 241 cgctagaagg tggggtgggg agagcagctg gactgggatg taagccatag caggactcca 301 cgagttgtca ctatcattta tcgagcacct actggtgtc cccagtgtcc tcagatctcc 361 atactgggg agccaggggc agcgacacgg tagctagccg tcgattggag aactttaaaa 421 tgaggactga attagctcat aaatggaaca cggcgcttaa ctgtgaggtt ggagcttaga 481 atgtgaaggg agaatgagga atgcgagact gggactgaga tggaccggc ggtgggagg 541 gggtgggggg atggaatttg aaccccggga gaggagatg gattttcta tggaggccga 601 cctgggatg gggagataag agagaccag gagggagtta atagggaat gggttgggg 661 cggcttgta aatgtgctgg gattaggctg tgcagata tgcaagg cttggaggc 721 taacctgggg tgaggccgggg gctggggtg tcactggtcc tcactggcgg 781 ttgattgaca gtttctcctt ccccagACTG GCCAATCACA GGCAGGAAGA TGAAGGTTCT 841 GTGGGCTGCG TTGCTGGTCA CATTCCTGGC AGgtatgggg gcggggcttg ctcggttccc 901 cccgctcctc cccctctcat cctcacctca acctcctggc cccattcagg cagaccctgg 961 gccccctctt ctgaggcttc tgtgctgctt cctggctctg aacagcgatt tgacgctctc 1021 tgggcctcgg tttcccccat ccttgagata ggagttagaa gttgttttgt tgttgttgtt 1081 tgttgttgtt gttttgtttt tttgagatga agtctcgctc tgtcgcccag gctggagtgc 1141 agtggcggga tctcggctca ctgcaagctc cgcctcccag gtccacgcca ttctcctgcc 1201 tcagcctccc aagtagctgg gactacaggc acatgccacc acacccgact aacttttttg 1261 tattttcagt agagacgggg tttcaccatg ttggccaggc tggtctggaa ctcctgacct 1321 caggtgatct gcccgtttcg atctcccaaa gtgctgggat tacaggcgtg agccaccgca 1381 cctggctggg agttagaggt ttctaatgca ttgcaggcag atagtgaata ccagacacgg 1441 ggcagctgtg atctttattc tccatcaccc ccacacagcc ctgcctgggg cacacaagga 1501 cactcaatac atgcttttcc gctgggcgcg gtggctcacc cctgtaatcc cagcactttg 1561 ggaggccaag gtgggaggat cacttgagcc caggagttca acaccagcct gggcaacata 1621 gtgagaccct gtctctacta aaaatacaaa aattagccag gcatggtgcc acacacctgt 1681 gctctcagct actcaggagg ctgaggcagg aggatcgctt gagcccagaa ggtcaaggtt 1741 gcagtgaacc atgttcaggc cgctgcactc cagcctgggt gacagagcaa gaccctgttt 1801 ataaatacat aatgctttcc aagtgattaa accgactccc ccctcaccct gcccaccatg 1861 gctccaaaga agcatttgtg gagcaccttc tgtgtgcccc taggtactag atgcctggac 1921 ggggtcagaa ggaccctgac ccaccttgaa cttgttccac acagGATGCC AGGCCAAGGT 1981 GGAGCAAGCG GTGGAGACAG AGCCGGAGCC CGAGCTGCGC CAGCAGACCG AGTGGCAGAG 2041 CGGCCAGCGC TGGGAACTGG CACTGGGTCG CTTTTGGGAT TACCTGCGCT GGGTGCAGAC 2101 ACTGTCTGAG CAGGTGCAGG AGGAGCTGCT CAGCTCCCAG GTCACCCAGG AACTGAGgtg 2161 agtgtcccca tcctggccct tgaccctcct ggtgggcggc tatacctccc caggtccagg 2221 tttcattctg cccctgtcgc taagtcttgg ggggcctggg tctctgctgg ttctagcttc 2281 ctcttcccat ttctgactcc tggctttagc tctctggaat tctctctctc agctttgtct 2341 ctctctcttc ccttctgact cagtctctca cactcgtcct ggctctgtct ctgtccttcc 2401 ctagctcttt tatatataga cagagagatg gggtctcact gtgttgccca ggctggtctt 2461 gaacttctgg gctcaagcga tcctcccgcc tcggcctccc aaagtgctgg gattagaggc 2521 atgagccacc ttgcccggcc tcctagctcc ttcttcgtct ctgcctctgc cctctgcatc 2581 tgctctctgc atctgtctct gtctccttct ctcggcctct gccccgttcc ttctctccct 2641 cttgggtctc tctggctcat ccccatctcg cccgcccat cccagccctt ctccccgcct 2701 cccactgtgc gacaccctcc cgccctctcg gccgcagGGC GCTGATGGAC GAGACCATGA 2761 AGGAGTTGAA GGCCTACAAA TCGGAACTGG AGGAACAACT GACCCCGGTG GCGGAGGAGA 2821 CGCGGGCACG GCTGTCCAAG GAGCTGCAGG CGGCGCAGGC CCGGCTGGGC GCGGACATGG 2881 AGGACGTGTG CGGCCGCCTG GTGCAGTACC GCGGCGAGGT GCAGGCCATG CTCGGCCAGA 2941 GCACCGAGGA GCTGCGGGTG CGCCTCGCCT CCCACCTGCG CAAGCTGCGT AAGCGGCTCC 3001 TCCGCGATGC CGATGACCTG CAGAAGCGCC TGGCAGTGTA CCAGGCCGGG GCCCGCGAGG 3061 GCGCCGAGCG CGGCCTCAGC GCCATCCGCG AGCGCCTGGG GCCCCTGGTG GAACAGGGCC 3121 GCGTGCGGGC CGCCACTGTG GGCTCCCTGG CCGGCCAGCC GCTACAGGAG CGGGCCCAGG 3181 CCTGGGGCGA GCGGCTGCGC GCGCGGATGG AGGAGATGGG CAGCCGGACC CGCGACCGCC 3241 TGGACGAGGT GAAGGAGCAG GTGGCGGAGG TGCGCGCCAA GCTGGAGGAG CAGGCCCAGC 3301 AGATACGCT GCAGGCCGAG GCCTTCCAGG CCCGCCTCAA GAGCTGGTTC GAGCCCCTGG 3361 TGGAAGACAT GCAGCGCCAG TGGGCCGGGC TGGTGGAGAA GGTGCAGGCT GCCGTGGGCA 3421 CCAGCGCCGC CCCTGTGCCC AGCGACAATC ACTGAACGCC GAAGCCTGCA GCCATGCGAC 3481 CCCACGCCAC CCCGTGCCTC CTGCCTCCGC GCAGCCTGCA GCGGGAGACC CTGTCCCCGC 3541 CCCAGCCGTC CTCCTGGGGT GGACCCTAGT TTAATAAAGA TTCACCAAGT TTCACGCA (SEQ ID NO: 230) where capital letters indicate exons in the APOE 3 pre-mRNA, and others indicate introns. This sequence contains the 5' untranslated region (5'UTR). The sequence was obtained from the University of California, Santa Cruz Genomic Institute Genome Browser under the identification number: hg38 ENST00000252486.9.

[0078] The present disclosure relates to oligonucleotides comprising about 18-20 nucleotides, wherein at least one nucleotide has a modification selected from the group consisting of bridged nucleic acids such as LNA, ENA, cET, 2'-fluoro modified nucleotides, 2O-methyl modified nucleotides, 2O-methoxy modified nucleotides, 2'-fluoroarabinonucleic acid (FANA), and combinations thereof, wherein the modification is located within a sequence of 5 nucleotides at the 5' and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes to an APOE transcript or mRNA of SEQ ID NO:1. The oligonucleotide hybridizes outside or inside the hybridization active region at, for example, positions 39, 41, 42, 77, 84, 124, 128, 130, 131, 204, -206, 208-214, 226-228, 295-296, 298-299, 307-309, 311, 339, 341-348, 447, 541-542, 550, 573-576, 580, 589, 592, 809, 812, 901-905, 1000-1004, 1006-1014, 1016-1017, 1019-1020, 1122-1123, and 1145-1147.

[0079] The present disclosure relates to oligonucleotides comprising about 14 to 18 nucleotides, wherein at least one nucleotide has a modification selected from the group consisting of a bridged nucleic acid (e.g., LNA, ENA, cET), a 2'-fluoro modified nucleotide, a 2O-methyl modified nucleotide, a 2O-methoxy modified nucleotide, a 2'-fluoroarabinonucleic acid (FANA), and combinations thereof, wherein the modification is located within a sequence of 5 nucleotides at the 5' and / or 3' end of the oligonucleotide, wherein the oligonucleotide hybridizes to an APOE transcript or pre-mRNA of SEQ ID NO: 230.Oligonucleotides include, for example, 45, 90, 127, 268, 269, 272, 273, 275, 276, 297, 298, 301, 319, 343, 344, 346, 347, 354, 380, 381, 384, 390, 391, 392, 394, 399, 400, 440, 444, 445, 446, 447, 448, 450, 451, 452, 453, 454, 456, 459, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531 64, 465, 485, 488, 489, 490, 492, 586, 769, 770, 788, 915, 916, 917, 919, 966, 967, 968, 969, 1005, 1006, 1007, 1008, 1009, 1036, 1037, 1327, 1328, 1329, 1330, 1331, 1333, 1334, 1335, 1336, 1386, 1424, 1425, 1491, 1725, 1726 26, 1727, 1728, 1746, 1823, 1855, 1856, 1857, 1875, 1898, 1899, 1900, 1902, 1904, 1905, 1906, 1925, 1950, 1951, 1985, 2148, 2150, 2151, 2211, 2233, 2234, 2235, 2236, 2260, 2261, 2347, 2348, 2355, 2356, 2357, 2358, 2359 , 2360, 2362, 2363, 2364, 2699, 2700, 2704, 2743, 2775, 2777, 2778, 2779, 2780, 2781, 3337, 3441, 3442, 3443, 3444, 3445, 3447, 3448, 3449, 3453, 3455, 3582, or 3583, or hybridizes outside or inside the hybridization active region.

[0080] Modified oligonucleotides complementary to APOE nucleic acids can be designed and tested for their effect on APOE mRNA in vitro. The modified oligonucleotides can be tested in a series of experiments under similar culture conditions.

[0081] The modified oligonucleotides in the table below can be uniformly modified oligonucleotides. The length of the oligonucleotides can be 21 nucleobases, and each nucleoside can have a 2'-substitution or modification described herein.

[0082] The modified oligonucleotides in Tables 1 and 2 below can be designed as gapmers. The gapmer can be 20 nucleosides in length, with a central gap segment containing 10 2'-deoxynucleosides flanked at both its 5' and 3' ends by wing segments, each containing 5 nucleosides (5-10-5 gapmer (except for ASO S1095 (SEQ ID NO: 81) which is a 4-10-4 gapmer), Table 1). Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment contains a 2'-modification. The 2'-modification is a 2'-methoxyethyl (e.g., 2'-MOE) modification.

[0083] In embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 2-1] [Table 2-2]

[0084] The modified oligonucleotides in Tables 3 and 4 below can be uniformly modified oligonucleotides. The length of the oligonucleotides can be 16 nucleobases, and each nucleoside can have a 2'-substitution or modification described herein.

[0085] The modified oligonucleotides in Tables 3 and 4 below can be designed as gapmers. The gapmer can be 16 nucleosides in length, with a central gap segment containing 10 2'-deoxynucleosides flanked at both its 5' and 3' ends by wing segments, each containing three nucleosides (a 3-10-3 gapmer). Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment contains a locked nucleic acid (LNA) modification.

[0086] In embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 4-1] [Table 4-2] [Table 5]

[0087] Example 1: Evaluation of Targeted Knockdown Efficacy and Toxicity of APOE-Specific ASOs in HEP3B Cells A detailed description of the methods used in this example can be found in the supplemental experimental section below. Briefly, the knockdown efficacy of 79 APOE-specific ASOs was evaluated in human HEP3B cells in vitro. Cells were treated with APOE-specific ASOs (12.5 nM) or non-silencing control oligonucleotides (12.5 nM) by transfecting them with LIPOFECTAMINE™ 2000 (THERMO FISHER SCIENTIFIC INC., 11668030). Forty-eight hours after treatment, cells were lysed. APOE and HPRT1 mRNA levels were analyzed using quantitative real-time polymerase chain reaction (qPCR), and APOE expression values ​​were normalized to the value of the endogenous housekeeping control gene HPRT1. The results of ASO-mediated APOE and HRPT1 knockdown in HEP3B cells are shown in Table 1 as residual APOE or HRPT1 mRNA expression compared with the non-silencing control ASO as a reference sample (normalized to 1).

[0088] Treating HEP3B cells with ASOs S1043, S1042, S1044, S1041, and S1025 (SEQ ID NOS: 11, 27-30) achieved >80% knockdown (represented by a residual APOE mRNA expression of <0.2 compared to cells treated with a non-targeting control ASO) (see Table 1). Of note, not all screened ASOs were highly efficient at downregulating APOE mRNA. Some ASOs, such as ASOs S1017, S1027, S1033, S1018, and S1065 (SEQ ID NOS: 3, 4, 13, 19, and 51), downregulated target gene expression by approximately 30% compared to non-targeting control ASOs. This indicates that our screening experiments identified target mRNA knockdown efficacies ranging from approximately 30% to approximately 90% of control levels among the 79 ASOs tested.

[0089] Changes in the expression levels of the HPRT1 housekeeping gene in response to ASO treatment (i.e., transfection) were used as a proxy to identify ASOs that exhibited cytotoxicity under the experimental conditions used. The use of the HPRT1 readout as an approximation of cytotoxicity was validated by live-cell imaging of HEP3B cell cultures. Figures 1A, 1B, and 1C show that cultures treated with ASO S1094 (SEQ ID NO: 80), which does not downregulate HPRT1 housekeeping gene expression (see Table 1), were nontoxic (Figure 1C), as determined by imaging, with confluency and cell morphology comparable to that of untreated cell cultures (Figure 1A). In contrast, cell cultures treated with ASOs that downregulate HPRT1 housekeeping gene expression (e.g., ASO S1038 (SEQ ID NO: 24)) (see Table 1) were not confluent with large numbers of cells but instead displayed abnormal morphology and density, pyknotic nuclei, indicative of cytotoxicity and cell death (Figure 1B). Since both ASOs S1094 (SEQ ID NO: 80) and S1038 (SEQ ID NO: 24) have comparable efficacy in downregulating the target gene APOE (see Table 1, approximately 70 to approximately 80% downregulation), the observed morphological changes are consistent with toxicity and HPRT1 mRNA is not associated with APOE downregulation.

[0090] Based on the observation that downregulation of the HPRT1 housekeeping gene coincided with confluency and cell morphology (e.g., indicators of microscopic cytotoxicity), the 79 screened molecules were further classified into two categories. Under the test conditions, ASOs that reduced HPRT1 mRNA by more than 20% in HEP3B cells were considered cytotoxic, while ASOs that modulated HPRT1 mRNA by less than 20% were considered non-toxic. Table 1 summarizes the HPRT1 mRNA expression levels after ASO transfection into HEP3B cells. ASOs S1024, S1088, S1047, S1077, and S1059 (SEQ ID NOs: 10, 33, 45, 63, and 74) resulted in robust APOE target inhibition (e.g., APOE mRNA knockdown) of >70% without any cytotoxic effects on HPRT1 housekeeping gene expression. In contrast, ASOs S1043, S1042, S1044, S1041, and S1025 (SEQ ID NOs: 11, 27, 28, 29, and 30) are highly effective in downregulating APOE mRNA but exhibit cytotoxic effects, reducing expression of the HPRT1 housekeeping gene by approximately 25% to approximately 40% in HEP3B cells.

[0091] In summary, we identified 37 ASOs (see Table 2) that down-regulate APOE target gene mRNA without causing significant cytotoxicity under our experimental conditions (e.g., no change in confluency or morphology compared to non-silencing controls) (see Figures 1A and 1C). Additional assays may be used to demonstrate the potency and efficacy and other properties of these molecules in vitro and in vivo.

[0092] Experimental Supplementary Section: This section provides details about the specific techniques and reagents used in the above example(s). Preparation of antisense oligonucleotides (ASOs): ASOs were synthesized by Integrated DNA Technologies (IDT) using their standard desalting process.

[0093] Cell culture: HEP3B cells (ATCC HB-8064) were maintained at 37°C and 5% CO in growth medium containing EMEM (ATCC 30-2003) with 10% FBS (VWR 97068-085) and 1% penicillin and 1% streptomycin (Pen-Strep) (THERMO FISHER SCIENTIFIC INC. 15140122) and passaged using 0.25% trypsin-EDTA (THERMO FISHER SCIENTIFIC INC. 25200-114) as a cell dissociation reagent. For ASO screening, HEP3B cells were plated at 15,000 cells per well of a 96-well plate in growth medium without Pen-Strep.

[0094] ASO transfection using Lipofectamine 2000: Each ASO was prepared as a 5 μM stock solution in ULTRAPURE™ DNase / RNase-Free Distilled Water (THERMO FISHER INC., 10977023) and stored at -80°C until use. Transfections were performed in triplicate culture wells as biological triplicates, with a final ASO concentration of 12.5 nM per well of a 96-well plate. The day after cell plating, the medium in each culture well was replaced with 90 μl of growth medium without Pen-Strep. For each ASO, the 5 μM stock solution (5 μM) was prediluted to 1.25 μM using ULTRAPURE™ DNase / RNase-Free Distilled Water. The prediluted ASO was further diluted to 0.25 μM using OPTI-MEM™ (THERMO FISHER INC., 31985062). LIPOFECTAMINE™ 2000 (THERMO FISHER SCIENTIFIC INC., 11668030) was prediluted to 6% (v / v) with OPTI-MEM™ and incubated for 5 minutes. Equal volumes (v / v) of 0.25 μM ASO in OPTI-MEM™ and 6% (v / v) LIPOFECTAMINE™ 2000 (THERMO FISHER SCIENTIFIC INC., 11668030) in OPTI-MEM™ were mixed and incubated for 10 minutes. 10 μl of the mixture was added to each culture well for a total volume of 100 μl per well. For the non-transfected control, all of the above steps were repeated except that 0.25 μM ASO in OPTI-MEM™ was replaced with OPTI-MEM™ alone. Cell confluency, as a surrogate observation marker for monitoring cell health and potential toxicity of ASO treatment, was determined by live-cell imaging of each well at 0, 24, and 48 hours post-transfection using the INCUCYTE® platform (SARTORIUS AG).

[0095] Cell lysis and quantitative RT-PCR: 48 hours after transfection, cells were lysed according to the instructions in the CELLS-TO-CT™ 1-step TAQMAN™ Kit (THERMO FISHER SCIENTIFIC, INC. A25602). 50 μl of RNA lysate was harvested per culture well and stored at -80°C until RT-qPCR. One-step RT-qPCR was performed in a 384-well plate. Each reaction consisted of 10 μl of a mixture containing 2.5 μl of TAQMAN™ 1-Step qRT-PCR Mix (THERMO FISHER SCIENTIFIC, INC.), 0.5 μl of APOE TAQMAN™ Assay FAM-MGB (Hs00171168_ml) (THERMO FISHER SCIENTIFIC, INC. 4331182), 0.5 μl of HPRT1 TAQMAN™ Assay VIC-MGB (Hs02800695_ml) (THERMO FISHER SCIENTIFIC, INC. 4448489), 1 μl of RNA lysate, and 5.5 μl of UltraPure™ DNase / RNase-Free Distilled Water. The thermal cycling method was as follows: 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles of i) 95°C for 3 seconds, ii) 60°C for 30 seconds. Cycle threshold (Ct) values ​​were analyzed using the delta-delta Ct method, with HRPT1 as the housekeeping gene and either a non-silencing control ASO or a non-transfection control as the reference sample. Variation between culture plates was controlled by including a non-transfection control, a non-silencing control ASO, and a positive control (S1087, SEQ ID NO: 73) identified from a pilot screen for APOE knockdown in the transfection scheme of each culture plate.Normalization between culture plates was performed using the "percentage of positive controls" method as described in Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R (2006) Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24:167-175.

[0096] Example 2: Evaluation of Targeted Knockdown Efficacy and Toxicity of APOE Pre-mRNA-Specific ASOs in HEP3B Cells A detailed description of the methods used in this example can be found in the supplemental experimental section below. Unless otherwise noted, all other methods used in this example were similar to those presented in Example 1 above. Briefly, the knockdown efficacy of 147 ASOs targeting APOE pre-mRNA was evaluated in human HEP3B cells in vitro. Cells were treated by delivering APOE pre-mRNA-targeting ASOs (5 μM) or the non-silencing control oligonucleotide S1250 (5 μM, SEQ ID NO: 83) without a carrier (gymnotic). Cells were lysed 72 hours after treatment. APOE and HPRT1 mRNA levels were analyzed using quantitative real-time polymerase chain reaction (qPCR). The results of APOE and HRPT1 knockdown by APOE pre-mRNA-targeting ASOs in HEP3B cells are shown in Table 3 as residual APOE or HRPT1 mRNA expression compared with the non-silencing control ASO as a reference sample (normalized to 1).

[0097] ASO S1104, S1105, S1108, S1110, S1111, S1114, S1116, S1120, S1123, S1124, S112 8, S1131, S1133, S1134, S1135, S1139, S1140, S1143, S1144, S1149, S1150, S11 51, S1153, S1154, S1155, S1156, S1158, S1170, S1171, S1177, S1183, S1184, S 1188, S1190, S1195, S1196, S1200, S1205, S1230, S1232, S1236, S1239, S1248, and S1249 (SEQ ID NOS: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229) (e.g., the ASOs in Table 4), achieved >30% knockdown (represented by residual APOE mRNA expression of <0.7 compared to cells treated with a non-targeting control ASO) (see Table 3). Treating HEP3B cells with ASOs S1104, S1139, S1140, S1143, S1144, S1149, S1153, S1154, S1155, S1156, S1188, S1205, S1248, and S1249 (SEQ ID NOs: 84, 119, 120, 123, 124, 129, 133, 134, 135, 136, 168, 185, 228, and 229) delivered without a carrier (gymnotic) achieved >50% knockdown (represented by residual APOE mRNA expression of <0.5 compared to cells treated with a non-targeting control ASO) (see Table 3). Several ASOs, S1144, S1153, S1154, S1156, and S1188 (SEQ ID NOs: 124, 133, 134, 136, and 168), resulted in a >60% reduction in HPRT1 mRNA compared to cells treated with a non-targeting control ASO.The lack of any visible cell death or morphological changes suggests experimental or biological variability in HPRT1 mRNA expression with carrier-free (gymnotic) ASO delivery.

[0098] Experimental Supplementary Section: This section describes the reagents, protocols, and details regarding the specific techniques used in Example 2.

[0099] Treatment with LNA-modified ASOs: Each ASO was prepared as a 500 μM stock solution in ULTRAPURE™ DNase / RNase-Free Distilled Water (THERMO FISHER INC., 10977023) and stored at -80°C until use. Treatment was performed in triplicate culture wells as biological triplicates, with a final ASO concentration of 5 μM per well of a 96-well plate. For each ASO, the 500 μM stock solution was prediluted to 250 μM using ULTRAPURE™ DNase / RNase-Free Distilled Water. One hour after cell plating, 2 μl of prediluted ASO was added to each culture well. Cell confluency, as a surrogate marker for monitoring cell health and potential toxicity of ASO treatment, was determined by live-cell imaging of each well 72 hours after treatment using the INCUCYTE® platform (SARTORIUS AG).

[0100] Cell lysis and quantitative RT-PCR: 72 hours after treatment (gymnotic delivery), cells were lysed according to the instructions in the CELLS-TO-CT™ 1-step TAQMAN™ Kit (THERMO FISHER SCIENTIFIC, INC. A25602). RNA lysates were harvested at 50 μl per culture well and stored at -80°C until RT-qPCR. One-step RT-qPCR was performed in a 384-well plate. Each reaction consisted of 10 μl of a mixture containing 2.5 μl of TAQMAN™ 1-Step qRT-PCR Mix (THERMO FISHER SCIENTIFIC, INC.), 0.5 μl of APOE TAQMAN™ Assay FAM-MGB (Hs00171168_ml) (THERMO FISHER SCIENTIFIC, INC. 4331182), 0.5 μl of HPRT1 TAQMAN™ Assay VICMGB (Hs02800695_ml) (THERMO FISHER SCIENTIFIC, INC. 4448489), 1 μl of RNA lysate, and 5.5 μl of UltraPure™ DNase / RNase-Free Distilled Water. The thermal cycling method was as follows: 50°C for 5 minutes, 95°C for 20 seconds, followed by 40 cycles of i) 95°C for 3 seconds, ii) 60°C for 30 seconds. Cycle threshold values ​​(Ct) were analyzed using the delta-delta Ct method, with HRPT1 as the housekeeping gene and either a non-silencing control ASO or treatment control as the reference sample. Variation between culture plates was controlled by including a non-silencing control ASO (S1250 (SEQ ID NO: 83)) and an assay control ASO for APOE knockdown (S1248 (SEQ ID NO: 228)) in each culture plate. Normalization between culture plates was performed using the "percentage of positive controls" method, as described in Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R (2006) Statistical practice in high-throughput screening data analysis. Nat Biotechnol 24:167-175.

[0101] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are those described herein. In describing and claiming the present invention, the following terminology will be used.

[0102] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0103] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0104] As used herein, "about" when referring to measurable values ​​such as amounts, lengths of time, and the like, is meant to encompass variations of 20% or 10%, more preferably 5%, even more preferably 1%, and even more preferably 0.1% from the specified value, and thus variations are applicable in the practice of the disclosed methods.

[0105] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, substance, or composition described herein that is effective in achieving a particular biological result or provides a therapeutic or prophylactic benefit.

[0106] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties that result therefrom. Thus, a gene encodes a protein when that protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually that presented in a sequence listing), and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding a protein or other product of that gene or cDNA.

[0107] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that versions of the nucleotide sequence that encode the protein may contain intron(s).

[0108] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, of which there are many types. Among other things, "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0109] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0110] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be construed as including all specifically disclosed possible subranges, as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed as including specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Claims

1. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a sequence that is at least 80% identical to SEQ ID NO:5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, or 77-81.

2. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a sequence selected from the group consisting of SEQ ID NOs: 5-11, 15, 16, 20-41, 43, 45, 47-50, 53, 55-57, 60-75, and 77-81.

3. 3. The antisense oligonucleotide of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the sequence is selected from the group consisting of SEQ ID NOs: 7, 8, 10, 15, 32, 33, 35, 36, 37, 38, 39, 40, 41, 43, 45, 49, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 71, 71, 72, 73, 74, 77, 78, 79, 80, and 81.

4. 4. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the antisense oligonucleotide or pharmaceutically acceptable salt thereof comprises from about 15 to about 25 nucleobases.

5. 5. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the antisense oligonucleotide or pharmaceutically acceptable salt thereof comprises from about 18 to about 22 nucleobases.

6. 6. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the antisense oligonucleotide comprises about 20 nucleobases.

7. 6. The antisense oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises about 18 nucleobases.

8. 10. The antisense oligonucleotide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more modified internucleoside linkages.

9. 9. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of claim 8, wherein the modified internucleoside linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesylphosphoramidate, and combinations thereof.

10. 9. The antisense oligonucleotide of claim 8, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

11. 9. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, wherein all of the internucleoside linkages are phosphorothioate linkages.

12. 10. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein the antisense oligonucleotide comprises DNA.

13. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the antisense oligonucleotide comprises RNA.

14. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the antisense oligonucleotide comprises a protein and / or a polypeptide.

15. 12. The antisense oligonucleotide of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises a small molecule.

16. 10. The antisense oligonucleotide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more modified nucleosides.

17. 17. The antisense oligonucleotide of claim 16, wherein the modified nucleoside is selected from the group consisting of a 2'-modified nucleoside, a 4'-modified nucleoside, a bridged nucleoside, a phosphorodiamidate morpholine, a locked nucleic acid, an ethylene-bridged nucleic acid, a glycol nucleic acid, a hexitol nucleic acid, a cyclohexene nucleic acid, an arabino nucleic acid, a peptide nucleic acid, a threose nucleic acid, a tricyclo-2'-deoxy-nucleotide, a 1'-deoxyribopentose, a 1',2'-dideoxyribopentose, a 2',3'-dideoxyribopentose, a 2',3'-didehydro-2',3'-dideoxyribopentose, a non-locked nucleic acid, and a combination thereof.

18. 18. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein the antisense oligonucleotide comprises one or more 2'-nucleoside modifications.

19. 19. The antisense oligonucleotide of claim 18, wherein the antisense oligonucleotide comprises at least eight 2'-nucleoside modifications.

20. 19. The antisense oligonucleotide of claim 18, wherein the antisense oligonucleotide comprises at least 10 2'-nucleoside modifications.

21. 19. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of claim 18, wherein all nucleosides of the antisense oligonucleotide contain 2'-nucleoside modifications.

22. 22. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 18 to 21, wherein the 2'-nucleoside modification is selected from the group consisting of a 2'-fluoro-nucleoside, a 2'-O-methyl-nucleoside, a 2'-O-methoxyethyl nucleoside, a 2'-O-benzyl-2'-deoxynucleoside, a 2'-O-methyl-4-pyridinyl nucleoside (2'-O-CH2Py(4)), a 2'-amino-nucleoside, and combinations thereof.

23. 10. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein said antisense oligonucleotide comprises one or more modified nucleobases.

24. 24. The antisense oligonucleotide of claim 23, or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is a methylated nucleobase.

25. 25. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 24, wherein the methylated nucleobase is selected from the group consisting of 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof.

26. 24. The antisense oligonucleotide of claim 23, or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is an acetylated nucleobase.

27. 27. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of claim 26, wherein the acetylated nucleobase is N4-acetylcytosine.

28. 24. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of claim 23, wherein the modified nucleobase is selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole-modified adenine, N-ethylpiperidine-6-triazole-modified adenine, 6-phenylpyrrolo-cytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine, and combinations thereof.

29. 10. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of the preceding claims, further comprising an inverted nucleotide.

30. 10. The antisense oligonucleotide or pharmaceutically acceptable salt thereof of any one of the preceding claims, wherein the antisense oligonucleotide is a gapmer.

31. 31. The antisense oligonucleotide of claim 30, wherein the gapmer comprises flanking RNA nucleotides at the 5' end, the 3' end, or both ends.

32. 32. The antisense oligonucleotide of claim 31, wherein the flanking RNA nucleotides at the 5'-end and / or 3'-end independently range from 1 nucleotide to about 9 nucleotides.

33. 33. The antisense oligonucleotide of any one of claims 30-32, wherein the gapmer ranges in length from about 18 to about 22 nucleosides.

34. The antisense oligonucleotide of any one of claims 30-32, wherein the gapmer is 18 nucleosides in length.

35. 35. The antisense oligonucleotide of any one of claims 30-34, wherein the gapmer comprises 10 central 2'-deoxynucleosides and 4 flanking RNA nucleotides at both the 5' and 3' ends.

36. The antisense oligonucleotide of any one of claims 30-33, wherein the gapmer is 20 nucleosides in length.

37. 37. The antisense oligonucleotide of any one of claims 30-33, or 36, wherein the gapmer comprises 10 central 2'-deoxynucleosides and / or 5 flanking ribonucleosides at both the 5' and 3' ends.

38. 10. The antisense oligonucleotide of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein administration of the antisense oligonucleotide to a subject reduces human APOE mRNA, pre-mRNA, protein expression, or a combination thereof.

39. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE mRNA is reduced by about 25% or more compared to the state before treatment.

40. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE mRNA is reduced by about 50% or more compared to the state before treatment.

41. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE mRNA is reduced by about 70% or more compared to the state before treatment.

42. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE protein is reduced by about 25% or more compared to the state before treatment.

43. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE protein is reduced by about 50% or more compared to the state before treatment.

44. 39. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 38, wherein human APOE protein is reduced by about 70% or more compared to the state before treatment.

45. 10. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein said antisense oligonucleotide is capable of hybridizing to human APOE mRNA.

46. 46. ​​The antisense oligonucleotide of claim 45, wherein the human APOE mRNA comprises a sequence that is at least 80% identical to SEQ ID NO:

1.

47. 46. ​​The antisense oligonucleotide of claim 45, wherein the human APOE mRNA encodes a protein comprising a sequence that is 85% identical to SEQ ID NO:

82.

48. 46. ​​The oligonucleotide of claim 45, wherein the APOE is an isoform selected from the group consisting of APOE2, APOE3, APOE4, APOE5f, APOE5s, and APOE7.

49. 46. ​​The oligonucleotide of claim 45, wherein the hybridization is by Watson-Crick base pairing.

50. 10. A method of treating a disease or disorder in a human subject in need thereof, said method comprising administering to a subject in need thereof an antisense oligonucleotide according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof.

51. 50. A method for treating a neurodegenerative disease, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof.

52. 52. The method of claim 51, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion diseases, and combinations thereof.

53. 52. The method of claim 51, wherein the neurodegenerative disease is Alzheimer's disease.

54. 52. The method of claim 51, wherein the neurodegenerative disease is a tauopathy.

55. 55. The method of claim 54, wherein the tauopathy is selected from the group consisting of primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral impairment frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

56. 50. A method of treating acute or chronic injury, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof.

57. 57. The method of claim 56, wherein the acute or chronic injury is selected from the group consisting of diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, anoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemobrain, neuronal damage resulting from tumors, meningitis, encephalitis, and other inflammation in the body or nervous system, or combinations thereof.

58. 50. A method of treating an acute or chronic inflammatory condition in the nervous system or in a body extremity, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof.

59. 59. The method of claim 58, wherein the acute or chronic inflammatory condition is selected from the group consisting of an autoimmune disease, stroke, multiple sclerosis, an infectious disease, an immune response to cancer, a tumor, or a combination thereof.

60. 50. A method of treating a vascular disease or vascular pathological condition, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof.

61. 61. The method of claim 60, wherein the vascular disease or vascular pathological condition is selected from the group consisting of any human vascular disease, including, but not limited to, cerebrovascular disease, cerebral microangiopathy, diseases caused by or affected by blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular disease, peripheral arterial disease, carotid artery disease, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency thrombosis, deep vein thrombosis, cerebral microangiopathy, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, or a combination thereof.

62. 50. A method of treating a lipid storage disorder, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof.

63. 63. The method of claim 62, wherein the lipid storage disorder is selected from the group consisting of cerebrotendinous xanthomatosis, Farber disease, Fabry disease, fucosidosis, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis AB variant, Krabbe disease, metachromatic leukodystrophy, multiple sulfatase deficiency, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sandhoff disease, Schindler disease, Tay-Sachs disease, Wolman disease, and combinations thereof.

64. 64. The method of any one of claims 50-63, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered locally to a subject in need thereof.

65. 65. The method of claim 64, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered orally, sublingually, intranasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, intracerebroventricularly, transdermally, and / or rectally.

66. 65. The method of claim 64, wherein the local administration is an intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

67. 64. The method of any one of claims 50-63, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need thereof.

68. 68. The method of any one of claims 50-67, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered in conjunction with one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents (both traditional chemotherapeutic agents and newer targeted therapy agents), radiation, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, costimulatory molecules, or combinations thereof.

69. 50. Use of the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof.

70. 50. Use of the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or disorder in a human subject in need thereof.

71. 50. Use of the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject in need thereof.

72. 50. Use of the antisense oligonucleotide of any one of claims 1-49, or a pharmaceutically acceptable salt thereof, for the treatment of a neurodegenerative disease in a subject in need thereof.

73. 73. The use of claim 71 or 72, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion diseases, and combinations thereof.

74. 73. The use of claim 71 or 72, wherein the neurodegenerative disease is Alzheimer's disease.

75. 73. The use of claim 71 or 72, wherein the neurodegenerative disease is a tauopathy.

76. 76. The use of claim 75, wherein the tauopathy is selected from the group consisting of primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral impairment frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

77. 1. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a sequence at least 80% identical to SEQ ID NO: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, or 229.

78. 1. An antisense oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 84, 85, 88, 90, 91, 94, 96, 100, 103, 104, 108, 111, 113, 114, 115, 119, 120, 123, 124, 129, 130, 131, 133, 134, 135, 136, 138, 150, 151, 157, 163, 164, 168, 170, 175, 176, 180, 185, 210, 212, 216, 219, 228, and 229, or a pharmaceutically acceptable salt thereof.

79. 79. The antisense oligonucleotide of claim 77 or 78, or a pharmaceutically acceptable salt thereof, wherein the sequence is selected from the group consisting of SEQ ID NOs: 127, 132, 147, 202, and 220.

80. 80. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 77-79, wherein the antisense oligonucleotide or pharmaceutically acceptable salt thereof comprises from about 12 to about 25 nucleobases.

81. 81. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 77-80, wherein the antisense oligonucleotide or pharmaceutically acceptable salt thereof comprises from about 14 to about 18 nucleobases.

82. 82. The antisense oligonucleotide of any one of claims 77-81, or a pharmaceutically acceptable salt thereof, wherein said antisense oligonucleotide comprises about 16 nucleobases.

83. 82. The antisense oligonucleotide of any one of claims 77-81, or a pharmaceutically acceptable salt thereof, wherein said antisense oligonucleotide comprises about 18 nucleobases.

84. 84. The antisense oligonucleotide of any one of claims 77-83, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more modified internucleoside linkages.

85. 85. The antisense oligonucleotide of claim 84, or a pharmaceutically acceptable salt thereof, wherein the modified internucleoside linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesylphosphoramidate, and combinations thereof.

86. 85. The antisense oligonucleotide of claim 84, or a pharmaceutically acceptable salt thereof, wherein said antisense oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

87. 85. The antisense oligonucleotide of claim 84, or a pharmaceutically acceptable salt thereof, wherein all of said internucleoside linkages are phosphorothioate linkages.

88. 88. The antisense oligonucleotide according to any one of claims 77 to 87, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises DNA.

89. 88. The antisense oligonucleotide according to any one of claims 77 to 87, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises RNA.

90. 88. The antisense oligonucleotide according to any one of claims 77-87, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises a protein and / or a polypeptide.

91. 88. The antisense oligonucleotide of any one of claims 77-87, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises a small molecule.

92. 92. The antisense oligonucleotide of any one of claims 77-91, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more modified nucleosides.

93. 93. The antisense oligonucleotide of claim 92, wherein the modified nucleoside is selected from the group consisting of 2'-modified nucleosides, 4'-modified nucleosides, bridged nucleosides, phosphorodiamidate morpholines, locked nucleic acids, ethylene-bridged nucleic acids, glycol nucleic acids, hexitol nucleic acids, cyclohexene nucleic acids, arabino nucleic acids, peptide nucleic acids, threose nucleic acids, tricyclo-2'-deoxy-nucleotides, 1'-deoxyribopentose, 1',2'-dideoxyribopentose, 2',3'-dideoxyribopentose, 2',3'-didehydro-2',3'-dideoxyribopentose, non-locked nucleic acids, and combinations thereof.

94. 94. The antisense oligonucleotide of any one of claims 77-93, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more 2'-nucleoside modifications.

95. 95. The antisense oligonucleotide of claim 94, wherein the antisense oligonucleotide comprises at least eight 2'-nucleoside modifications.

96. 95. The antisense oligonucleotide of claim 94, wherein the antisense oligonucleotide comprises at least 10 2'-nucleoside modifications.

97. 95. The antisense oligonucleotide of claim 94, or a pharmaceutically acceptable salt thereof, wherein all nucleosides of the antisense oligonucleotide comprise 2'-nucleoside modifications.

98. The antisense oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 94 to 97, wherein the 2'-nucleoside modification is selected from the group consisting of a 2'-fluoro-nucleoside, a 2'-O-methyl-nucleoside, a 2'-O-methoxyethyl nucleoside, a 2'-O-benzyl-2'-deoxynucleoside, a 2'-O-methyl-4-pyridinyl nucleoside (2'-O-CH2Py(4)), a 2'-amino-nucleoside, and combinations thereof.

99. 99. The antisense oligonucleotide of any one of claims 77-98, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

100. 100. The antisense oligonucleotide of claim 99, or a pharmaceutically acceptable salt thereof, wherein said modified nucleobase is a methylated nucleobase.

101. The antisense oligonucleotide of claim 100, or a pharmaceutically acceptable salt thereof, wherein the methylated nucleobase is selected from the group consisting of 5-methyluracil, N6-methyladenine, N4-methylcytosine, N7-methylguanine, 5-hydroxymethylcytosine, N3-methylcytosine, and combinations thereof.

102. 100. The antisense oligonucleotide of claim 99, or a pharmaceutically acceptable salt thereof, wherein said modified nucleobase is an acetylated nucleobase.

103. 103. The antisense oligonucleotide of claim 102, or a pharmaceutically acceptable salt thereof, wherein said acetylated nucleobase is N4-acetylcytosine.

104. 100. The antisense oligonucleotide of claim 99, or a pharmaceutically acceptable salt thereof, wherein the modified nucleobase is selected from the group consisting of pseudouridine (Ψ), N1-methyl-pseudouridine (N1-methyl-Ψ), 2-thiouridine (s2U), 5-fluoro-2'-deoxyuridine (FUDR), 8-oxo-7,8-dihydroguanosine (8-oxoG), N-ethylpiperidine-7-EAA triazole-modified adenine, N-ethylpiperidine-6-triazole-modified adenine, 6-phenylpyrrolo-cytosine (PhpC), 2,4-difluorotoluyl-ribonucleoside (rF), N1(5-nitroindole)ribonucleoside, 5-methoxyuridine, and combinations thereof.

105. 105. The antisense oligonucleotide of any one of claims 77-104, or a pharmaceutically acceptable salt thereof, further comprising an inverted nucleotide.

106. 106. The antisense oligonucleotide or pharmaceutically acceptable salt of any one of claims 77-105, wherein the antisense oligonucleotide is a gapmer.

107. 107. The antisense oligonucleotide of claim 106, wherein the gapmer comprises flanking RNA nucleotides at the 5' end, the 3' end, or both ends.

108. 108. The antisense oligonucleotide of claim 107, wherein the flanking RNA nucleotides at the 5'-end and / or 3'-end independently range from about 1 nucleotide to about 5 nucleotides.

109. The antisense oligonucleotide of any one of claims 106-108, wherein the gapmer ranges in length from about 14 to about 22 nucleosides.

110. The antisense oligonucleotide of any one of claims 106-108, wherein the gapmer is 18 nucleosides in length.

111. The antisense oligonucleotide of any one of claims 106-110, wherein the gapmer comprises 10 central 2'-deoxynucleosides and 3 flanking RNA nucleotides at both the 5' and 3' ends.

112. The antisense oligonucleotide of any one of claims 106-109, wherein the gapmer is 16 nucleosides in length.

113. 113. The antisense oligonucleotide of any one of claims 106-109, or 112, wherein the gapmer comprises 10 central 2'-deoxynucleosides and / or 3 flanking ribonucleosides at both the 5' and 3' ends.

114. The antisense oligonucleotide of any one of claims 77-113, or a pharmaceutically acceptable salt thereof, wherein administration of the antisense oligonucleotide to a subject reduces human APOE mRNA, pre-mRNA, protein expression, or a combination thereof.

115. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE pre-mRNA or mRNA is reduced by about 10% or more compared to the state before treatment.

116. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE pre-mRNA or mRNA is reduced by about 30% or more compared to the state before treatment.

117. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE pre-mRNA or mRNA is reduced by about 50% or more compared to the state before treatment.

118. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE protein is reduced by about 25% or more compared to the state before treatment.

119. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE protein is reduced by about 50% or more compared to the state before treatment.

120. 115. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 114, wherein human APOE protein is reduced by about 70% or more compared to the state before treatment.

121. 121. The antisense oligonucleotide according to any one of claims 77-120, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is capable of hybridizing to human APOE pre-mRNA.

122. 122. The antisense oligonucleotide of claim 121, wherein the human APOE pre-mRNA comprises a sequence that is at least 80% identical to SEQ ID NO:

230.

123. 122. The antisense oligonucleotide of claim 121, wherein the human APOE pre-mRNA encodes a protein comprising a sequence that is 85% identical to SEQ ID NO:

82.

124. 122. The oligonucleotide of claim 121, wherein the APOE is an isoform selected from the group consisting of APOE2, APOE3, APOE4, APOE5f, APOE5s, and APOE7.

125. 122. The oligonucleotide of claim 121, wherein the hybridization is by Watson-Crick base pairing.

126. 126. A method of treating a disease or disorder in a human subject in need thereof, said method comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof.

127. 126. A method for treating a neurodegenerative disease, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125 or a pharmaceutically acceptable salt thereof.

128. 128. The method of claim 127, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion diseases, and combinations thereof.

129. 128. The method of claim 127, wherein the neurodegenerative disease is Alzheimer's disease.

130. 128. The method of claim 127, wherein the neurodegenerative disease is a tauopathy.

131. 131. The method of claim 130, wherein the tauopathy is selected from the group consisting of primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral disorder-type frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

132. 126. A method for treating acute or chronic injury, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof.

133. 133. The method of claim 132, wherein the acute or chronic injury is selected from the group consisting of diffuse axonal injury, concussion, edema, hematoma, skull fracture, hemorrhage, hypoxic brain injury, anoxic brain injury, carbon monoxide poisoning, cerebral aneurysm, stroke, hydrocephalus, traumatic brain injury, spinal cord injury, chronic traumatic encephalopathy, chemobrain, neuronal damage resulting from tumors, meningitis, encephalitis, and other inflammation in the body or nervous system, and combinations thereof.

134. 126. A method of treating an acute or chronic inflammatory condition in the nervous system or in a body extremity, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof.

135. 135. The method of claim 134, wherein the acute or chronic inflammatory condition is selected from the group consisting of an autoimmune disease, stroke, multiple sclerosis, an infectious disease, an immune response to cancer, a tumor, and combinations thereof.

136. 126. A method of treating a vascular disease or vascular pathological condition, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof.

137. 137. The method of claim 136, wherein the vascular disease or vascular pathological condition is selected from the group consisting of any human vascular disease, including, but not limited to, cerebrovascular disease, cerebral microangiopathy, diseases caused by or affected by blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, peripheral vascular disease, peripheral arterial disease, carotid artery disease, pulmonary embolism, abdominal aortic aneurysm, collagen vascular disease, chronic venous insufficiency thrombosis, deep vein thrombosis, cerebral microangiopathy, blood-brain barrier leakage, atherosclerosis, cerebral amyloid angiopathy, and combinations thereof.

138. 126. A method of treating a lipid storage disorder, comprising administering to a subject in need thereof the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof.

139. 139. The method of claim 138, wherein the lipid storage disorder is selected from the group consisting of cerebrotendinous xanthomatosis, Farber disease, Fabry disease, fucosidosis, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis AB variant, Krabbe disease, metachromatic leukodystrophy, multiple sulfatase deficiency, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Sandhoff disease, Schindler disease, Tay-Sachs disease, Wolman disease, and combinations thereof.

140. 140. The method of any one of claims 126-139, wherein said oligonucleotide or a pharmaceutically acceptable salt thereof is administered locally to a subject in need thereof.

141. The method of claim 140, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered orally, sublingually, intranasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, intracerebroventricularly, transdermally, and / or rectally.

142. 141. The method of claim 140, wherein the local administration is an intracranial, intracerebral, intramuscular, spinal, epidural, sacroiliac, or subdural injection.

143. 140. The method of any one of claims 126-139, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered systemically to a subject in need thereof.

144. 144. The method of any one of claims 126-143, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof is administered in conjunction with one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents (both traditional chemotherapeutic agents and newer targeted therapy agents), radiation, kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, costimulatory molecules, or combinations thereof.

145. 126. Use of the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof.

146. 126. Use of the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or disorder in a human subject in need thereof.

147. 126. Use of the antisense oligonucleotide of any one of claims 77-125, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject in need thereof.

148. 126. Use of the antisense oligonucleotide according to any one of claims 77 to 125, or a pharmaceutically acceptable salt thereof, for the treatment of a neurodegenerative disease in a subject in need thereof.

149. 149. The use of claim 147 or 148, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, dementia with Lewy bodies, multiple sclerosis, multiple system atrophy, Parkinson's disease, prion diseases, and combinations thereof.

150. 149. The use of claim 147 or 148, wherein the neurodegenerative disease is Alzheimer's disease.

151. 149. The use of claim 147 or 148, wherein the neurodegenerative disease is a tauopathy.

152. 152. The use of claim 151, wherein the tauopathy is selected from the group consisting of primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia and parkinsonism linked to chromosome 17, vacuolar tauopathy, Litiko-Bodig disease, ganglioglioma, gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis, lead encephalopathy, tuberous cerebral sclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, behavioral disorder-type frontotemporal dementia (Pick's disease), argyrophilic grain disease, and combinations thereof.

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